Impact of microencapsulated Ziziphora tenuior essential oil and orange fiber as natural‐functional additives on chemical and microbial qualities of cooked beef sausage

Abstract The aim of the current study was to investigate the suitability of Ziziphora tenuior essential oil (ZEO) as a preservative. For this purpose, the effect of free and microencapsulated ZEO, combined with orange fiber, was determined on the chemical and microbial qualities of cooked beef sausage. In this study, modified starch was used for encapsulation of essential oil, and subsequently, 0.5% ZEO and 1% orange fiber were used for preparing cooked beef sausages during 60 days of storage at 4°C. To assess the microbial quality of samples, total viable count (TVC), psychrophilic count (PSY), and lactic acid bacteria (LAB) were analyzed. Furthermore, peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) were tested to examine lipid oxidation. The most components of ZEO were pulegone (47.12%), isomenthone (14.57%), and 1,8‐cineole (12.84%) according to GC–MS analysis. The reducing power, DPPH radical scavenging activity, MIC, and MBC of ZEO were 16.44 (EC50), 8.36 (IC50), 0.625–2.5, and 1.25–5 mg/ml, respectively. Moreover, sausage containing 0.5% microencapsulated ZEO in combination with 1% orange fiber showed the best results with the following values (p ≤ .05): TVC (3.69 log CFU/g), PSY (3.51 log CFU/g), LAB (3.1 log CFU/g), PV (10.41 meq/kg lipid), and TBARS (3.1 mg MDA/kg). This is due to the antimicrobial and antioxidant properties of microencapsulated essential oil. Therefore, the results of the present study can be applied in the meat industries as a new natural preservation method.


| INTRODUC TI ON
Meat and meat products, such as sausages, deem as important food products with a great deal of socioeconomic impacts. Regrettably, meat products are categorized as perishable foods due to their rapid spoilage and safety challenges. The main factors causing spoilage of sausage and reducing its shelf life are microbial growth and lipid oxidation (Luong et al., 2020;Zehi et al., 2020). Microbial contamination of meat and meat products is frequently reported by legal authorities (EFSA & ECDC, 2019). In addition, lipid oxidation is regarded as one of the most important criteria for meat and meat product quality (Bolívar-Monsalve et al., 2019). In recent years, in order to control the spoilage of these products, the formulation and production of novel processed meat, with functional properties and without chemical preservatives, has been one of the main priorities for the research and development section of meat industries to respond to the green marketing and consumerism demand (Alirezalu et al., 2021;Pereira et al., 2019).
Essential oils (EOs) are natural compounds, proved to have antimicrobial and antioxidant activity (Mohajer et al., 2021), and have been widely used in processed meats due to their beneficial effects (Šojić et al., 2021;Tomović et al., 2020). Addition of Eos to sausages formulation improves their microbial and chemical stability and safety (Šojić et al., 2021;Viuda-Martos et al., 2010a). Microencapsulation of Eos has progressed remarkably in recent years and could provide several benefits, including controlled release, reduced volatility, increased availability, solubility, stability, and bioavailability (increased antimicrobial and antioxidant activities; Abd Manaf et al., 2015;Viacava et al., 2018). Encapsulation of EOs can increase the durability and antimicrobial and antioxidant strengths of EOs in the sausage samples due to the slow release of EOs (Ji et al., 2022;Vafania et al., 2019).
Ziziphora tenuior is a plant belonging to the Lamiaceae family, and its EO is recognized as an aromatic compound with remarkable antioxidant and antimicrobial activities (Behravan et al., 2007). The antimicrobial properties of Z. tenuior have been investigated against Escherichia coli, Staphylococcus aureus, and Shigella dysenteriae (Mahboubi et al., 2014), as well as its antioxidant properties (Dakah et al., 2014).
Different dietary fibers have been added to food formulas, as an ingredient, to produce new functional foods such as meat products (Díaz-Vela et al., 2017), chicken nuggets (Mohd Zaini et al., 2021), and dairy products (Yi et al., 2014). The advantages of the addition of fibers to foods can be divided into two categories: (a) promote foods' properties such as improving the texture and color, reducing sugar and fat content, and enhancing antioxidant activity; (b) beneficial effects on human health including cardiovascular health and weight management (Viuda-Martos et al., 2010b). Herein, the addition of fiber to foods has become an upward trend in the market (Sloan, 2000).
Various researches have investigated the use of the free form of different EOs in combination with different fibers on the chemical and microbial stability of meat and meat products (Aminzare et al., 2018;Araújo et al., 2018;Bianchin et al., 2017;da Silva et al., 2018;Huang et al., 2021;Meira et al., 2017;Mohammadpourfard et al., 2021;Viuda-Martos et al., 2010a, 2010b, while as far as we know, there are no previous studies evaluating antimicrobial and antioxidant effects of EOs, in microencapsulated and free forms, in combination with fibers. Therefore, the objectives of the current work were to (a) examine the total phenolic content and chemical composition of ZEO, (b) determine the in vitro antimicrobial and antioxidant activities of ZEO, and (c) evaluate the effects of ZEO, in free and microencapsulated forms, alone and in combination with orange fiber on microbial and oxidative stability of cooked beef sausage stored at 4°C during 60 days.

| Extraction of essential oil
Ziziphora tenuior herb was gathered from Zanjan province, Iran (spring 2020), and a senior taxonomist confirmed the plant species.
Cold tap water was used to wash the aerial section of the herb and placed at 25°C to become dry. A mixer device (ParsKhazar, Tehran, Iran) was used to ground dry herbs. By using a Clevenger apparatus (KOL, behr, Düsseldorf, Germany) ZEO was extracted (hydrodistillation method). The extraction process was continued for about 180 min at 100°C. Sodium sulfate was used for dehydration of oil and subsequently filtered through a 0.22 μm filter. Yielded EO was transferred to a dark, sealed glass container and placed at 4°C away from direct light, until use (Hamedi et al., 2017).

| ZEO gas chromatography-mass spectrometry (GC-MS) analysis
A Hewlett Packard 5890 device (using HP-5MS column; the dimension of the film was 30 × 0.25 mm ID × 0.25 mm) was used to analyze the chemical composition of ZEO. For the beginning, the device temperature was set at 50°C; it increased 2°C per min to reach 120°C and remained at 120°C for 3 min. The temperature was raised to 300°C, and the helium flow rate during this process was 1 ml/min. Also, ionization energy was 70 eV for the MS process.
Subsequently, retention indices were compared with samples presented by the library (Wiley-VCH2001 data software, Weinheim, Germany).

| Analysis of total phenolic content
Folin-Ciocalteu reagent assay was used to measure the amount of total phenolic compounds of ZEO (Aliakbarlu et al., 2013;Singleton et al., 1999). An amount of 2.25 ml of distilled water and 500 μl of EO in methanol mixture (4 mg/ml) were transferred to a glass tube.
Folin-Ciocalteu reagent (250 μl) was then added to the tube and mixed vigorously. Two milliliters of sodium carbonate solution (7.5%; w/v) was added and mixed, 5 min after the addition of the reagent.
The tube was then placed at 25°C for 120 min, and by using a spectrophotometer (CECIL, Cambridge, UK), at 760 nm, absorbance was recorded. Five hundred microliter 50% (v/v) methanol was used as blank. The same procedure was also applied to a standard solution of gallic acid, and a standard curve was obtained. The content of total phenolic was shown as mg GAE/g oil.

DPPH free radical scavenging activity
The DPPH free radical scavenging method was used to investigate the antioxidant properties of ZEO and reported as IC 50 values. For this purpose, 1 ml of ZEO with different concentrations (0.312-10 mg/ml) was transferred separately to a glass tube and DPPH methanolic solution (1 ml, 90 μM). Methanol was added to the tube to reach the final volume of 4 ml. The glass tube was shaken well and incubated at room temperature for 1 h, away from direct light.
Using a spectrophotometer (CECIL) at 517 nm, the absorbance was measured against a blank. For each concentration, radical scavenging activity was figured by the following equation: Abs DPPH : methanolic solution of DPPH absorbance Abs sample : ZEO absorbance In this test, the positive control was BHT. The concentration of the sample was represented by IC 50 values. Based on the graph, for IC 50 , it was needed to scavenge half of DPPH free radicals, which was plotted for inhibition percentage (Guleria et al., 2013).

Ferric (Fe3+) reducing power
Ziziphora tenuior essential oil was analyzed to determine its ferric reducing power introduced by Guleria et al. (2013). Potassium ferricyanide (2.5 ml, 1%) and phosphate buffer (2.5 ml, 0.2 M, pH 6.6) were added to 1 ml of each ZEO concentrations (0.312-10 mg/ ml) in a glass tube and placed in an incubator at 50°C for 20 min.
Subsequently, trichloroacetic acid (2.5 ml, 10%) was transferred to the glass tube. Centrifugation of the mixture was carried out for 10 min at 1000 g. The supernatant (2.5 ml) was transferred to another tube, 2.5 ml ferric chloride solution (1%) and 2.5 ml distilled water were mixed with supernatant, and the absorbance of tube solution was recorded with a double beam ultraviolet-VIS spectrophotometer at 700 nm. Higher reducing power displays higher absorbance. The graph of absorbance was used for the calculation of 0.5 of absorbance (EC 50 ). EC 50 of ZEO compared with standard antioxidant and positive control in this analysis was BHT.

Preparation of bacteria
In this study, 10 common foodborne pathogens (Listeria monocy- Fifteen milliliters of BHI broth was used to activate bacteria (24 h, at 37°C; Ojagh et al., 2010). Centrifugation was then carried out, and bacterial cells were washed two times with normal saline. The concentration of each bacterium was adjusted by a spectrophotometer (CECIL) at 600 nm to the required density (~1.5 × 10 8 CFU/ ml) and dilution was carried out to achieve proper bacterial density (~1.5 × 10 6 CFU/ml) for further evaluation. For confirmation of the results, bacteria were cultivated in BHI agar and placed in an incubator for 24 h at 37°C.

Agar disk diffusion
By using the agar disk diffusion method, ZEO was analyzed for antimicrobial activity against 10 aforementioned bacteria (Goni et al., 2009). Briefly, 100 μl of overnight grown bacteria (~1.5 × 10 6 CFU/ml) was cultivated on nutrient agar. Sterile filter paper in the shape of disks, containing 10 μg/disk of ZEO, was then placed onto the surface of culture media and were placed into an incubator for 24 h at 37°C, to allow bacterial growth. Blank disk (with no antibacterial agent) and disks containing ampicillin (10 μg/ disk) were used as negative and positive controls, respectively.
Growth inhibition of bacteria was determined by measuring the inhibition zone diameters of surrounding disks by caliper (Zaidan et al., 2005).

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The MIC values for ZEO were investigated according to the study of Aliakbarlu et al. (2013). Briefly, 20 μl of various concentrations of Radical scavenging activity ( % ) = Abs DPPH − Abs sample ∕ Abs DPPH × 100. ZEO (3.125-100 mg/ml) in DMSO 5% as well as 20 μl of each bacterial suspension and 160 μl of BHI broth were transferred into 96well microplates. The final concentration of ZEO was in the range of 0.312-10 mg/ml; each well was reached to 200 μl final volume, containing ~1.5 × 10 5 CFU/ml bacteria. Positive control had culture medium and suspension of bacteria and the negative control contained the medium and ZEO. A microplate shaker was used to mix the plates (for about 30 s, at 300 g) and finally placed in an incubator for 24 h at 37°C. The minimum concentration of ZEO with no bacterial growth (without turbidity) was considered as MIC. Wells with no visible growth were then cultured on BHI agar and placed in an incubator at 37°C for 24 h. The 99.9% bacterial death caused by the lowest ZEO concentration was considered as MBC (Araújo et al., 2018).

| Preparation of microencapsulated ZEO
Ziziphora tenuior essential oil was microencapsulated according to the method previously described by Baranauskienė et al. (2007)) with some modifications. Modified starch (30% w/w) was added to 40°C deionized water and dispersed. The solution was then cooled and mixed for 12 h to increase hydration. Ziziphora tenuior essential oil (10% w/w) was added to hydrated coating starch and emulsified.
An Ultra Turrax homogenizer (IKA T25 basic, Staufen, Germany) was utilized for homogenization, functioned at 13,500 g for 7 min. The emulsion was spray-dried in a mini spray dryer (Buchi B191, Flawil, Gallen, Switzerland) under the following parameters: The emulsion inlet flow was 15 ml/min and inlet and outlet temperature of air were 180 ± 10 and 90 ± 10°C, respectively. Yielded powder was placed at −18°C freezer until use.

| Sausage formulation
The sausage was prepared in the Dara meat industry, Tehran, Iran.
Mortadella-type sausage was produced according to a traditional and commercial formula using 50% beef meat, 18% ice, 16% vegetable oil, 10% flour, 3% potato starch, 1.2% sodium chloride, 0.7% garlic, 0.6% spice mix (black pepper, nutmeg, and ginger), 0.05% sodium ascorbate, 0.012% sodium nitrate, and 0.5% sodium tripolyphosphate. They were then subjected to the cutter and homogenized thoroughly. The batter was then divided into five equal portions, and experimental groups were prepared using free or microencapsulated ZEO (the final concentration in the product was 0.5% w/w of pure ZEO) alone and in combination with 1% (w/w) orange fiber (Table 1). Herein, the minimum effective concentration of ZEO and orange fiber was used in order to avoid undesirable sensory effects on sausage sample (Fernández-López et al., 2008;Rezaei & Shahbazi, 2018). Subsequently, they were packed into polyamide bags (50 mm caliber; IranNavid, Tehran, Iran) weighed 50 ± 1 g, closed and sealed at both ends, placed in a cooking room, monitored by a thermocouple probe (Omega Engineering, Stamford, CT, USA), and ensured that sausage center reached to 75°C. The sausage was placed in a water bath to cool down and subsequently at 4°C until analysis. Analysis was carried out at 0, 10, 20, 40, and 60 days of storage.

| Microbiological analysis
Major microbiological quality indicators of sausages were evaluated during the storage period. Total psychrophilic bacteria count (PSY), total viable count (TVC), and lactic acid bacteria (LAB) were investigated for the microbiological quality of sausages. Briefly, 90 ml sterile peptone water (0.1%) and 10 g of sample were placed in a sterile bag and homogenized using a stomacher (Interscience, St. Nom, France) for 1 min. Subsequently, serial dilutions were prepared in glass tubes, and diluted samples were transferred to appropriate culture media. The microbiological analysis was carried out with respect to the study of Esmaeili et al. (2020). For TVC, 0.1 ml of proper dilution was transferred to PCA medium and placed in an incubator for 24 h at 37°C. A similar method was used for PSY, but plates were stored for 10 days at 7°C. To perform LAB count, 1 ml of diluted samples was poured into sterile plates, and 15-20 ml of MRS agar was added to the plates. The plates were then gently rotated and incubated anaerobically (anaerobic jars with GasPak system type C) at 30°C for 72 h. The mean was reported as log CFU/g (Nisar et al., 2019). The peroxide value of the extracted total lipid from sausage samples was analyzed, similar to the method previously illustrated by Lekjing (2016). Thirty milliliter chloroform: acetic acid (2:3) and 1 g lipid sample were added to a plastic container and shaken vigorously. Then, saturated potassium iodide solution (0.5 ml) was poured into the container and held at room temperature protected from light, for 1 min.
Subsequently, distilled water (30 ml) was added to the container. The indicator (starch solution, 0.5 ml, 1% w/v) was transferred to the mixture and standardized. Sodium thiosulfate solution (0.01 N) was used for the titration of iodine. The values were expressed as meq/kg lipid.

Thiobarbituric acid reactive substances (TBARS)
Ten grams of beef sausage samples were homogenized with extraction solution (35 ml, containing 1 ml BHT [5 mg/ml] and 4% perchloric acid) for 1 min. The solution was filterate through a Whatman filter paper and collected in a 50 ml falcon tube. By adding 4% perchloric acid, the filtrate was reached to 50 ml, and TBA solution (5 ml, 0.02 mol/L) and the adjusted mixture (5 ml) was poured into a glass tube. Subsequently, the glass tube was then shaken by a vortex mixer (Lab Genius, London, UK) and placed in a water bath (100°C) for 1 h for the formation of malonaldehyde-TBA complex. A spectrophotometer (CECIL) was used to measure sample absorbance at 532 nm. The amounts of TBARS were shown as mg MDA/kg. TEP solution was utilized to prepare standard curve (Pikul et al., 1989).

| Statistical analysis
In this study, the entire tests were performed in technical triplicates, and the results were statistically analyzed using SPSS software (SPSS Statistics Software, version 18). The data were tested by using one-way ANOVA and Tukey's test. p ≤ .05 was considered as a statistical significance level.

| ZEO chemical analysis
The components of essential oils exhibit antimicrobial and antioxi- other studies, which was in accordance with the current study (Baser et al., 1991;Behravan et al., 2007;Ozturk & Ercisli, 2007). However, some other main components reported were different; for example, in the study of Behravan et al. (2007), other main constituents of ZEO were terpineol (14.5%) and methyl acetate (10.9%). Besides, Ajourloo et al. (2021) reported geraniol (20.62%) and carvacrol (18.17%) as the major constituents for ZEO, which was inconsistent with the current study. These variations in the major components of EOs attributed to environmental factors, genetic factors such as cultivar, extraction method, maturity of the plants, the solvent used for extraction, geographical location, the part used of plant, the season of harvest, and cultivation conditions (Hazrati et al., 2020).
Moreover, it has been proven that pulegone is effective against some bacteria, making it suitable for microbial growth in food models (Damani et al., 2022).

| In vitro antioxidant activities of ZEO
The results of antioxidant activities of ZEO were measured by reducing power and DPPH free radical scavenging methods, which are shown in

| In vitro antibacterial activity of ZEO
In vitro antibacterial analysis of ZEO showed inhibition for growth patterns against pathogens (Tables 4 and 5 Overall, the data demonstrated that ZEO was effective against Gram-positive bacteria more than Gram-negative ones. This is in compliance with the study of Hamedi et al. (2017), which investigated the antibacterial activity of Ziziphora persica EO and reported lower MIC and MBC for Gram-positive bacteria than Gram-negatives. In addition, other studies also indicated reasonable antibacterial effect of ZEO (Hazrati et al., 2020;Nazemisalman et al., 2018). It has been suggested that the existence of a major layer of lipopolysaccharide and lipoprotein in the cell wall of these bacteria (Gram-negative) makes them resistant to ZEO, while the thin layer of Gram-positive bacteria is their weakness against EOs such as Ziziphora species EOs (Mahdavi et al., 2020).

| Microbiological analysis of cooked sausage
TVC, PSY, and LAB of cooked sausage for different treatments during 60 days of storage are outlined in Figures 1, 2, and 3, respectively.
At the beginning of the storage period, no bacteria were detected in all groups, and similar results were observed on day 10 of storage.  (6.71 log CFU/g), while the lowest amount was measured for MZEOF (3.69 log CFU/g). Similar results were measured on day 60 for PSY analysis (control: 5.81 and MZEOF: 3.51 log CFU/g). With regard to LAB, the highest bacterial count in day 60 was recorded for the control group (6.09 log CFU/g), and the lowest was for the MZEOF treatment (3.10 log CFU/g).
The results showed lower values for ZEOF in comparison with the ZEO group at day 60, indicating a significant effect of fiber addition on microbial counts of sausage (p ≤ .05). Moreover, according to ISIRI (standards institute of Iran), the maximum acceptable limit for TVC in cooked sausages is set at 5 log CFU/g (ISIRI, 2005). MZEO and MZEOF groups were the only samples that did not exceed this limit

| Evaluation of lipid oxidation in cooked sausage
In this study, PV test was done to evaluate the oxidation of lipids in sausage samples, and the constant increasing trend was observed for this analysis. Peroxide value is one of the most common analyses for the evaluation of primary oxidation in foods.

| CON CLUS ION
In this study, 0.5% free or microencapsulated ZEO, alone and in combination with 1% orange fiber, was added to the formulation of cooked beef sausage, and their impacts on microbial (TVC, PSY, and LAB) and chemical (PV and TBARS) qualities of the product were assessed. The initial evaluation of ZEO composition and properties showed acceptable potential for application in the sausage. The major constituent of ZEO was pulegone (47.12%), followed by isomenthone (14.57%).
The data obtained by microbiological and lipid oxidation corroborated that all treatment groups were able to maintain the properties of sausage. Considering this, the best results were observed in the sausage samples treated with 0.5% microencapsulated ZEO and 1%